Project Summary Glaucoma leads to a progressive loss of retinal ganglion cells by mechanisms that are not fully understood. At present, lowering the intraocular pressure (IOP) is the only treatment, and new therapeutic approaches that prevent ganglion cell degeneration in a manner independent of IOP would be welcome. There is evidence that the first signs of ganglion cell degeneration occur in the optic nerve head where the ganglion cell axons exit the globe through a hole in the sclera to form the optic nerve. In this region, the axons are unmyelinated and come into direct contact with astrocytes. Optic nerve astrocytes react to injury ? such as an increase in IOP - with changes in their morphology and gene expression pattern. We showed that, at least in the early stages of the disease, astrocyte reactivity is a protective response and preventing it leads to a worse outcome for ganglion cells and visual function. This result suggests that astrocytes, or astrocyte-derived factors, can be harnessed for a neuroprotective glaucoma therapy that could be added to IOP-lowering drugs that are already in use. In our search for astrocyte-derived factors that mediate the protective response, we identified secreted phosphoprotein 1 (SPP1, also called osteopontin). This protein is expressed only at low levels in the normal optic nerve, but it is robustly up-regulated in all rodent glaucoma models studied so far. In addition, SPP1 is constitutively expressed in a sparse population of retinal ganglion cells. Using an SPP1 knock out mouse, we showed that SPP1 deficiency leads to morphological signs of astrocyte and axon damage in the optic nerve head even in the absence of elevated IOP. In the microbead occlusion model of glaucoma, SPP1 deficient mice lose more ganglion cells and have worse visual function than wild-type controls. Most importantly, virus- mediated overexpression of SPP1 in the retina is highly protective of ganglion cell function and prevents ganglion cell loss without affecting IOP. Based on these findings, we believe that SPP1 may be a promising candidate for a neuroprotective therapy in glaucoma. However, at present we do not know whether the SPP1 expression in reactive astrocytes or in retinal ganglion cells or both are needed to achieve optimal protection. To address this question, we have designed a transgenic mouse strain that will allow for cell-type specific deletion of SPP1 in astrocytes, retinal ganglion cells (or other cell types) separately. The mice will also express different fluorescent marker proteins in targeted cells before and after the deletion of SPP1. We will use this new tool to address our hypothesis that astrocyte-derived SPP1 in the optic nerve and ganglion cell-derived SPP1 in the retina are both necessary for optimal ganglion cell protection in glaucoma (Aims 1 and 2). In our translational Aim 3, we will test whether overexpression of SPP1 is protective of retinal ganglion cell function in the long term and assess ocular tissues for adverse effects that may result from chronic SPP1 overexpression.